Isolation and characterization of a novel extracellular metalloprotease from Bacillus subtilis.

We have isolated and characterized two minor extracellular proteases from culture supernatants of a strain of Bacillus subtilis containing deletion mutations of the genes for the extracellular proteases subtilisin (apr) and neutral protease (npr) and a minor extracellular protease (epr) as well as intracellular serine protease-I (isp-1). Characterization studies have revealed that one of these enzymes is the previously described protease bacillopeptidase F. The second enzyme, the subject of this report, is a novel metalloprotease, which we designate Mpr. Mpr is a unique metalloprotease that has been purified to apparent homogeneity by using both conventional and high-performance liquid chromatography procedures. Mpr has a molecular mass of approximately 28 kilodaltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a basic isoelectric point of 8.7. The enzyme showed maximal activity against azocoll at pH 7.5 and 50 degrees C. Mpr was inhibited by dithiothreitol and a combination of beta-mercaptoethanol and EDTA. Activity was moderately inhibited by beta-mercaptoethanol and EDTA alone as well as by cysteine and citrate and only marginally by phosphoramidon 1,10-phenanthroline and N-[N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine. Mpr was not inhibited by phenylmethylsulfonyl fluoride. In addition, Mpr showed esterolytic but not collagenolytic activities. Our studies suggest that Mpr is a secreted metalloprotease containing cysteine residues that are required for maximal activity.     

Gene encoding a novel extracellular metalloprotease in Bacillus subtilis.

The gene for a novel extracellular metalloprotease was cloned, and its nucleotide sequence was determined. The gene (mpr) encodes a primary product of 313 amino acids that has little similarity to other known Bacillus proteases. The amino acid sequence of the mature protease was preceded by a signal sequence of approximately 34 amino acids and a pro sequence of 58 amino acids. Four cysteine residues were found in the deduced amino acid sequence of the mature protein, indicating the possible presence of disulfide bonds. The mpr gene mapped in the cysA-aroI region of the chromosome and was not required for growth or sporulation.     

Increased production of extracellular enzymes by the synergistic effect of genes introduced into Bacillus subtilis by stepwise transformation.

The amyR3, amyS, papS1, tmr, and papM118 mutations each stimulate alpha-amylase production two- to sevenfold above the level in a wild-type strain of Bacillus subtilis. A strain which presumably has all five of these mutations produced 250-fold more alpha-amylase.     

Cloning and characterization of Bacillus subtilis iep, which has positive and negative effects on production of extracellular proteases.

We have isolated a DNA fragment from Bacillus subtilis 168 which, when present in a high-copy plasmid, inhibited production of extracellular alkaline and neutral proteases. The gene responsible for this activity was referred to as iep. The open reading frame of iep was found to be incomplete in the cloned DNA fragment. When the intact iep gene was reconstructed after the missing part of the iep gene had been cloned, it showed an enhancing effect on the production of the extracellular proteases. The open reading frame encodes a polypeptide of 229 amino acids with a molecular weight of ca. 25,866. Deletion of two amino acids from the N-terminal half of the putative iep protein resulted in dual effects, i.e., a decrease in the inhibitory activity shown by the incomplete iep gene and a slight increase in the enhancing activity shown by the complete iep gene. These results show that the iep gene product is a bifunctional protein, containing inhibitory and enhancing activities for the exoprotease production in the N-terminal and C-terminal regions, respectively. It was found by genetic and functional analyses that iep lies very close to sacU.     

Cloning and characterization of a gene of Streptomyces griseus that increases production of extracellular enzymes in several species of Streptomyces.

Summary A 7.2 kbBglII restriction fragment, which increases the production of several extracellular enzymes, including alkaline phosphatase, amylase, protease, lipase and -galactosidase, was cloned inStreptomyces lividans from the DNA ofS. griseus ATCC 10137. This gene (namedsaf) showed a positive gene dosage effect on production of extracellular enzymes. When thesaf gene was introduced into cells in high copy numbers it delayed the formation of pigments and spores inS. lividans and also retarded actinorhodin production inStreptomyces coelicolor. Thesaf gene hybridized with specific bands in the DNA of severalStreptomyces strains tested. A 1 kb fragment containing thesaf gene was sequenced and contains an open reading frame (ORF) of 306 nucleotides which encodes a polypeptide of M_r 10 500. This ORF is contained within a fragment of 432 by which retained activity inStreptomyces. A fragment with promoter activity is present upstream of thesaf reading frame. The predicted Saf polypeptide has a strong positive charge, and does not show a typical amino acid composition for a membrane protein, and contains a DNA-binding domain similar to those found in several regulatory proteins.     

Cloning and expression of a novel protease gene encoding an extracellular neutral protease from Bacillus subtilis.

We have cloned from Bacillus subtilis a novel protease gene (nprB) encoding a neutral protease by using a shotgun cloning approach. The gene product was determined to have a molecular mass of 60 kDa. It has a typical signal peptide-like sequence at the N-terminal region. The expression of nprB can be stimulated by using a B. subtilis strain, WB30, carrying a sacU(h)h mutation. Expression of this protease gene results in production of a 37-kDa protease in the culture medium. The first five amino acid residues from the N terminus of the mature protease were determined to be Ala-Ala-Gly-Thr-Gly. This indicates that the protease is synthesized in a preproenzyme form. The purified protease has a pH optimum of around 6.6, and its activity can be inhibited by EDTA, 1,10-phenanthroline (a zinc-specific chelator), and dithiothreitol. It retained 65% of its activity after treatment at 65 degrees C for 20 min. Sequence comparison indicates that the mature form of this protease has 66% homology with the two thermostable neutral proteases from B. thermoproteolyticus and B. stearothermophilus. It also shares 65, 61, and 56% homology with the thermolabile neutral proteases from B. cereus, B. amyloliquefaciens, and B. subtilis, respectively. The zinc-binding site and the catalytic residues are all conserved among these proteases. Sequence homology extends into the "propeptide" region. The nprB gene was mapped between metC and glyB and was not required for growth or sporulation.     

Cloning and characterization of the gene for an additional extracellular serine protease of Bacillus subtilis.

We have purified a minor extracellular serine protease from a strain of Bacillus subtilis bearing null mutations in five extracellular protease genes: apr, npr, epr, bpr, and mpr (A. Sloma, C. Rudolph, G. Rufo, Jr., B. Sullivan, K. Theriault, D. Ally, and J. Pero, J. Bacteriol. 172:1024-1029, 1990). During purification, this novel protease (Vpr) was found bound in a complex in the void volume after gel filtration chromatography. The amino-terminal sequence of the purified protein was determined, and an oligonucleotide probe was constructed on the basis of the amino acid sequence. This probe was used to clone the structural gene (vpr) for this protease. The gene encodes a primary product of 806 amino acids. The amino acid sequence of the mature protein was preceded by a signal sequence of approximately 28 amino acids and a prosequence of approximately 132 amino acids. The mature protein has a predicted molecular weight of 68,197; however, the isolated protein has an apparent molecular weight of 28,500, suggesting that Vpr undergoes C-terminal processing or proteolysis. The vpr gene maps in the ctrA-sacA-epr region of the chromosome and is not required for growth or sporulation.     

Increased production of extracellular enzymes by the synergistic effect of genes introduced into Bacillus subtilis by stepwise transformation

The amyR3, amyS, papS1, tmr, and papM118 mutations each stimulate alpha-amylase production two- to sevenfold above the level in a wild-type strain of Bacillus subtilis. A strain which presumably has all five of these mutations produced 250-fold more alpha-amylase.     

Cloning and characterization of the Bacillus subtilis prkA gene encoding a novel serine protein kinase

We have cloned and sequenced a 3574-bp Bacillus subtilis (Bs) DNA fragment located between the nrdA and citB genes at about 169° on the chromosome. An Escherichia coli strain, LBG1605, carrying a mutated ptsH gene (encoding HPr (His-containing protein) of the bacterial phosphotransferase system (PTS)) and complemented for PTS activity with the ptsH of Staphylococcus carnosus, exhibited reduced mannitol fermentation activity when transformed with a plasmid bearing this 3574-bp Bs fragment. This fragment contained an incomplete and two complete open reading frames (ORFs). The product of the first complete ORF, a protein composed of 235 amino acids (aa) (25 038 Da), was found to be responsible for the observed reduced mannitol fermentation. The 3′ part of this 705-bp second ORF and the 428-bp incomplete first ORF encode aa sequences exhibiting almost 40% sequence identity. However, the function of these two proteins remains unknown. The third ORF, the 1893-bp prkA gene, encodes a protein (PrkA) of 72 889 Da. PrkA possesses the A-motif of nucleotide-binding proteins and exhibits distant homology to eukaryotic protein kinases. Several of the essential aa in the loops known to form the active site of cyclic adenosine 3′,5′-monophosphate (cAMP)-dependent protein kinase appeared to be conserved in PrkA. After expression of prkA and purification of PrkA, we could demonstrate that PrkA can indeed phosphorylate a Bs 60-kDa protein at a Ser residue.     

Proton motive force may regulate cell wall-associated enzymes of Bacillus subtilis.

Bacterial metabolism excretes protons during normal metabolic processes. The protons may be recycled by chemiosmosis, diffuse through the wall into the medium, or bind to cell surface constituents. Calculations by Koch (J. Theor. Biol. 120:73-84, 1986) have suggested that the cell wall of gram-positive bacteria may serve as a reservoir of protons during growth and metabolism, causing the wall to have a relatively low pH. That the cell wall may possess a pH lower than the surrounding medium has now been tested in Bacillus subtilis by several independent experiments. When cultures of B. subtilis were treated with the proton conductors azide and carbonylcyanide m-chlorophenylhydrazone, the cells bound larger amounts of positively charged probes, including the chromium (Cr3+) and uranyl (UO2(2+) ions and were readily agglutinated by cationized ferritin. In contrast, the same proton conductors caused a decrease in the binding of the negatively charged probe chromate (CrO4(2-)). Finally, when levansucrase was induced in cultures by the addition of sucrose, the enzyme was inactive as it traversed the wall during the first 0.7 to 1.0 generation of growth. The composite interpretation of the foregoing observations suggests that the wall is positively charged during metabolism, thereby decreasing its ability to complex with cations while increasing its ability to bind with anions. This may be one reason why some enzymes, such as autolysins, are unable to hydrolyze their substrata until they reach the wall periphery or are in the medium.     

Transfer and expression of mutations in pleiotropic genes determining the synthesis of extracellular enzymes in Bacillus subtilis

The mutations in the genes determining the levels of extracellular enzymes production were transferred into Bacillus subtilis 168 strain by genetic transformation technique. The method used has permitted registering the transfer of pleiotropic genes. Seven amylase producers were detected among 126 his+ transformants screened, as well as five metalprotease producers among 246 gly+ transformants. Cotransfer of pap and hpr mutant genes linked with his+ or gly+ genes might result in finding the producers among the mentioned prototrophic transformants. Selection of linked markers in transformation, presented in the paper, is discussed to be a useful technique for obtaining producer strains for industrial production.     

Cloning and characterization of Bacillus subtilis homologs of Escherichia coli cell division genes ftsZ and ftsA.

The Bacillus subtilis homolog of the Escherichia coli ftsZ gene was isolated by screening a B. subtilis genomic library with anti-E. coli FtsZ antiserum. DNA sequence analysis of a 4-kilobase region revealed three open reading frames. One of these coded for a protein that was about 50% homologous to the E. coli FtsZ protein. The open reading frame just upstream of ftsZ coded for a protein that was 34% homologous to the E. coli FtsA protein. The open reading frames flanking these two B. subtilis genes showed no relationship to those found in E. coli. Expression of the B. subtilis ftsZ and ftsA genes in E. coli was lethal, since neither of these genes could be cloned on plasmid vectors unless promoter sequences were first removed. Cloning the B. subtilis ftsZ gene under the control of the lac promoter resulted in an IPTGs phenotype that could be suppressed by overproduction of E. coli FtsZ. These genes mapped at 135 degrees on the B. subtilis genetic map near previously identified cell division mutations.